The overall objective of this research is to understand the structural basis for the production of a functional cytochrome P450 (P450) molecule. The emphasis of this proposal is on a) targeting to the endoplasmic reticulum (ER) mediated by the signal anchor sequence (SA), and b) interaction of the catalytic domain with the ER membrane. The specific aims of the proposal are to first, identify and characterize accessory proteins that are involved in the direct ER retention of P450, with emphasis on BAP31 which has been shown to interact with P450 and mediate ER retention. Second, examine whether the position and orientation of the N-terminal signal anchor sequence in the membrane, determined by cysteine accessibility, correlates with the ER retention function;third, determine if P450 2C2, but not P450 2E1, is excluded from the protein exit domains of the ER by analysis of colocalization with protein exit markers by fluorescence microscopy and by immuno-enrichment of the exit domain;fourth, determine whether BAP31 increases P450 levels by functioning as an ER retention protein or by targeting P450 for degradation by examining effects of specific protease inhibitors on P450 levels and distribution;fifth, determine whether the F-G loop region of P450 2C8 is part of a dimerization interface as in the crystallized protein by disulfide linkage of cysteines substituted in the interface sequences or if the F-G loop region is inserted into the lipid bilayer by cysteine accessibility assays, and sixth, determine if the hydrophobicity of membrane-contacting loops of the catalytic domain correlates with activity of the P450s by introducing mutations that reduce hydrophobicity. Affinity purification and two-hybrid approaches will be used to identify proteins interacting with P450, bimolecular fluorescence complementation and fluorescence emission resonance transfer will be used to monitor interactions, and siRNA or knock-out ES cells will be used for functional analysis of potential ER retention proteins. P450s mediate the detoxification of many drugs and toxins, activation of carcinogens and pro- drugs, and biogenesis of many endogenous compounds. Mutations in human P450s have dramatic clinical effects on drug metabolism and steroid biogenesis. Understanding the genetic and structural basis for generating functional P450s is necessary to understand the role of these enzymes in normal and pathological states.